Use of remote ischemic conditioning for traumatic injury

ABSTRACT

The invention provides methods for reducing traumatic injury through the use of ischemic conditioning.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/322070, filed on Apr. 8, 2010, entitled “USE OF REMOTE ISCHEMICCONDITIONING FOR TRAUMATIC INJURY”, the entire contents of which areincorporated by reference herein.

BACKGROUND OF THE INVENTION

Traumatic injury is one of the leading causes of death in children andyoung adults. In addition, it represents a significant proportion ofhealth care expenditures. Trauma generally refers to severe bodilyinjury or wounding, typically resulting from blunt force as can occurwith falls, automobile accidents, domestic or industrial accidents, andin battle. Traumatic injuries should be treated immediately in order toprevent or lessen the effects of such injuries. Many of theinterventions and/or therapies known to be useful at least in the acutestages of traumatic injury however are not always available.

SUMMARY OF THE INVENTION

The invention is premised, in part, on the surprising finding thatdeliberately performing one or more cycles of transient blood flowocclusion followed by reperfusion in a region of the body of a subject,that is remote from the region where trauma occurs or that is beingprotected from the effects of the trauma, reduces the effects of suchtrauma. The trauma may be direct trauma or indirect trauma. The traumamay or may not be associated with hypovolemia. The methods of theinvention provide benefit even if resuscitation therapy is performed,and more surprisingly even if resuscitation therapy is delayed.

The invention therefore provides for the use of remote ischemicconditioning (RIC) to treat traumatic injury. The invention contemplatesthe use of RIC on a subject that has experienced, is experiencing, or islikely to experience trauma. In subjects that have experienced or areexperiencing trauma, the invention calls for RIC during or after thetrauma, or during and after the trauma. In subjects that are likely toexperience trauma, the invention calls for RIC before the expectedtrauma and optionally during and/or after such trauma occurs.

Accordingly, in one aspect, the invention provides a method for reducingor preventing injury to cells, tissues, or organs of a body as a resultof trauma comprising performing one or more cycles of blood flowocclusion followed by reperfusion on a subject before, during, and/orfollowing trauma, wherein the blood flow occlusion and reperfusionoccurs at a remote region of the body, including but not limited to oneor more lower and/or upper limbs. It will be understood that the bloodflow occlusion and reperfusion cycles will be performed in an amount andfrequency to reduce or prevent injury. In another aspect, the inventionprovides a method for reducing or preventing injury to cells, tissues,or organs of a body as a result of trauma comprising performingindividual or repeated RIC regimens on a subject before, during, and/orfollowing trauma. It will be understood that the RIC regimens will beperformed in an amount and a frequency sufficient to reduce or preventinjury.

In some embodiments, the injury affects multiple organs, and may bereferred to as multi-organ injury or dysfunction. In some embodiments,the injury is to the lungs and/or to the liver. In some embodiments, theinjury is neurological injury or dysfunction.

In another aspect, the invention provides a method for treating asubject experiencing trauma comprising performing one or more cycles ofblood flow occlusion followed by reperfusion on the subject duringand/or following trauma, wherein the blood flow occlusion andreperfusion occurs at a remote region of the body, including but notlimited to one or more lower and/or upper limbs. In another aspect, theinvention provides a method for treating a subject experiencing traumacomprising performing individual or repeated RIC regimens on the subjectduring and/or following trauma.

In another aspect, the invention provides a method comprising performingone or more cycles of blood flow occlusion followed by reperfusion on asubject experiencing or likely to experience trauma, wherein the one ormore cycles are performed before, during and/or following trauma,wherein the blood flow occlusion and reperfusion occurs at a remoteregion of the body, including but not limited to one or more lowerand/or upper limbs. In another aspect, the invention provides a methodcomprising performing individual or repeated RIC regimens on the subjectexperiencing or likely to experience trauma, wherein the one or more RICregimens are performed before, during and/or following trauma.

The invention contemplates that the subject will undergo one or morethan one RIC regimen. Such multiple regimens may be performed on asingle day and/or over the course of several days.

In some embodiments, the trauma is hemorrhagic shock. in someembodiments, the subject may be hypotensive and/or hypovolemic. In someembodiments, the subject has lost 10%, 15%, 20%, 25%, 30%, or more oftotal whole blood volume. In some embodiments, the subject has receivedresuscitation therapy. In some embodiments, the subject has not receivedresuscitation therapy.

In some embodiments, the trauma is not associated with hypovolemia(e.g., the trauma may be blast injury).

In sonic embodiments, the repeated RIC regimens comprise more than oneRIC regimen performed on a single day. In some embodiments, the repeatedRIC regimens comprise two, three, four or five RIC regimens performed ona single day. In some embodiments, the repeated RIC regimens compriseone or more RIC regimens on more than one day.

In some embodiments, at least one RIC regimen is performed within 30minutes or 1 hour of the trauma. In some embodiments, at least one RICregimen is performed immediately after trauma.

In some embodiments, the repeated RIC regimens comprise one or more RICregimens performed on a daily basis for one month.

In some embodiments, the RIC regimens are performed before the trauma,in which case they may be regarded as remote ischemic preconditioningregimens. In some embodiments, the RIC regimens are performed before andafter the trauma. In some embodiments, the RIC regimens are performedbefore, during and after the trauma. In some embodiments, the RICregimens are performed during and after the trauma. In some embodiments,the RIC regimens are performed after the trauma.

In some embodiments, the subject is human.

In some embodiments of the foregoing aspects, a single RIC cycle isperformed on the subject (i.e., the subject is subjected to a period ofblood flow occlusion followed by a period of reperfusion). In someembodiments, the cycle comprises about 10 minutes of blood flowocclusion and about or less than 10 minutes of reperfusion.

In some embodiments, at least one individual RIC regimen (for example,within the repeated RIC regimen) comprises at least two, at least three,or at least four cycles, each cycle comprising blood flow occlusion andreperfusion. In some embodiments, at least one RIC regimen comprises atleast four cycles, each cycle comprising blood flow occlusion andreperfusion. In some embodiments, at least one RIC regimen comprises oneor more cycles of about 5 minutes of blood flow occlusion and about 5minutes of reperfusion. In some embodiments, at least one RIC regimencomprises one or more cycles of about 10 minutes of blood flow occlusionand about 10 minutes of reperfusion. In some embodiments, blood flowocclusion is effected by applying to the remote region of the body apressure that is above systolic pressure. in some embodiments, bloodflow occlusion is effected by applying to the remote region of the bodya pressure that is below systolic pressure.

In some embodiments, at least one individual RIC regimen (for example,within the repeated RIC regimen) comprises at least two, at least three,or at least four cycles, each cycle comprising supra-systolic pressureand reperfusion. In some embodiments, at least one RIC regimen comprisesat least four cycles, each cycle comprising supra-systolic pressure andreperfusion. In some embodiments, at least one RIC regimen comprises oneor more cycles of 5 minutes of supra-systolic pressure and 5 minutes ofreperfusion. In some embodiments, the supra-systolic pressure is apressure that is 1-5 mmHg, or 1-10 mmHg, or 1-15 mmHg above systolicpressure. in some embodiments, the supra-systolic pressure is a pressurethat is at least 15 mmHg above systolic pressure. In some embodiments,the supra-systolic pressure is pressure that is about 200 mmHg.

In some embodiments, at least one individual RIC regimen (for example,within the repeated RIC regimen) comprises at least two, at least three,or at least four cycles, each cycle comprising below-systolic pressureand reperfusion.

In some embodiments, individual or repeated RIC regimens are performedat same site. In one embodiment, individual or repeated RIC regimens areperformed on an upper limb. In one embodiment, the individual orrepeated RIC regimens are performed on a lower limb. In one embodiment,individual or repeated RIC regimens are performed using two or moredevices such as two or more cuffs, positioned at different sites on thebody (e.g., one cuff per upper limb (or arm), two cuffs on a singleupper limb, one cuff per lower limb (or leg), two cuffs on a singlelower limb, one cuff on an upper limb and one cuff on a lower limb,etc.).

In various embodiments, the subject may be administered two or more ofthese aforementioned agents.

These and other aspects and embodiments of the invention will bediscussed in greater detail herein.

BRIEF DESCRIPTION OF THE FIGURES

lire accompanying figures are not intended to be drawn to scale. In thefigures, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in every figure.

Various embodiments of the invention will now be described, by way ofexample, with reference to the accompanying figures, in which:

FIG. 1 is a schematic representation of one embodiment of a remoteischemic conditioning system, including a pneumatically inflatable cuffconfigured to contract about the limb of a subject.

FIG. 2 is a block diagram of one embodiment of an operating scheme ofthe RIC system.

FIG. 3 shows an alternate embodiment of a cuff configured to contractabout the limb of a subject.

FIGS. 4A and 4B show the effect of RIC on shock and resuscitationinduced liver damage in individual mice (FIG. 4A) and as an average(FIG. 4B).

FIGS. 5A and 5B show the effect of RIC on shock and resuscitationinduced serum TNF-alpha levels in individual mice (FIG. 5A) and as anaverage (FIG. 5B).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides, in part, methods for treating subjects that haveexperienced trauma by performing on such subjects one or more RICregimens during and/or after the trauma. The invention also provides, inpart, methods for reducing the effects of trauma in subjects likely toexperience trauma by performing one or more RIC regimens prior totrauma. Although not intending to be bound by any mechanism or theory ofaction, it is contemplated that the one or more RIC cycles or regimensreduce the degree of injury in cells, one or more tissues and/or one ormore organs that would be impacted by the trauma. RIC regimens are ableto ameliorate the effects of ischemia and reperfusion injury whenperformed before or during the time of ischemia or reperfusion. RIC isassociated with suppression of immune mediators believed to beresponsible for trauma-associated morbidity.

The invention relates to the performance of RIC to treat (including toameliorate) the systemic effects associated with traumatic injury.Examples of traumatic injury that can he treated according to theinvention include but are not limited to blunt trauma and hemorrhage(e.g., hemorrhagic shock). In sonic aspects of the invention, a subjectthat has sustained a traumatic injury will undergo one or more cycles ofdeliberately induced blood flow occlusion and reperfusion at a locationon the body that is uninjured (e.g., an uninjured limb). In some aspectsof the invention, a subject that has sustained a traumatic injury willundergo one or more cycles of deliberately induced ischemia andreperfusion at a location on the body that is uninjured (e.g., anuninjured limb). As described in greater detail below, these cyclesconstitute a regimen. A regimen may comprise 1, 2, 3, 4, 5 or morecycles. Each cycle comprises induced occlusion or ischemia for a firstperiod of time followed by reperfusion for a second period of time. Thefirst and second periods of time may be identical or they may bedifferent from each other. As a non-limiting example, one or more cyclesof a regimen may comprise about 5 minutes of occlusion or ischemiafollowed by about 5 minutes of reperfusion. As another non-limitingexample, one or more cycles may comprise about 10 minutes of occlusionor ischemia followed by about 10 minutes of reperfusion. If only asingle cycle is used, then the reperfusion period may be shorter thanthe occlusion or ischemic period (e.g., it may be less than about 1minute, less than about 30 seconds, less than about 10 seconds, etc.).

The invention contemplates that RIC will be performed on the subject bya first responder (i.e., the first qualified person to attend to thesubject). RIC can therefore be performed using an automated device (suchas a pressure cuff) or manually (using a tourniquet). The ability toachieve therapeutic benefit using RIC alone is invaluable incircumstances where other interventions, including intravenous fluidresuscitation, are not available or are delayed. These circumstancesinclude without limitation battlefield conditions during militaryconflicts. Accordingly, the invention contemplates that RIC can be usedto reduce and/or prevent injury that is induced by trauma (e.g.,hemorrhagic shock) in situations in which resuscitation therapy has notbeen performed or was delayed. RIC may be performed before the trauma,before resuscitation therapy, and/or after the resuscitation therapy.The invention provides intervention in the form of a light-weight,preferably automated, ischemic conditioning device that can be performedby any personnel with minimal training and minimal time required fortreatment. In like manner, the invention also contemplates performingRIC on a subject in preparation for a probable traumatic injury,including for example prior to military engagement or confrontation.

Accordingly, the degree and/or severity of traumatic injury can bereduced by deliberately and repeatedly performing cycles of inducedtransient ischemia and reperfusion (i.e., an RIC regimen) in subjects.These subjects include those that are experiencing trauma and those thatare likely to experience trauma. The ease of use of these methods makethem amenable to particular circumstances including but not limited tobattlefield injuries. The ability to provide therapy to such subjects,particularly where there is no other therapy or intervention immediatelyavailable, is valuable. In like manner, these methods can be used inother emergency situations in which no other therapy or intervention isimmediately available such as can occur following catastrophic eventssuch as earthquakes and other natural disasters, bombings, and the like.

The invention contemplates, in some aspects, performing a repeated RICregimen on a subject. As used herein, an RIC regimen (or art individualRIC regimen) means at least one cycle of an induced transient ischemicevent followed by a reperfusion event. An individual RIC regimentherefore may be comprised of 1, 2, 3, 4, 5, or more such cycles.

Also as used herein, a repeated RIC regimen is two or more individualRIC regimens that occur on a single day and/or one or more RIC regimensthat occur on a number of days. For example, the repeated RIC regimenmay comprise performing multiple RIC regimens on a single day, orperforming single RIC regimens on a number of days, or performingmultiple RIC regimens on a number of days. If the repeated RIC regimenoccurs on a single day, the time between individual regimens may be atleast 10 minutes, at least 20 minutes, at least 40 minutes, at least 1hour, at least 2 hours, or at least 6 hours, for example. The inventioncontemplates that more than one RIC regimen may be performed prior to anexpected trauma (such as a battlefield injury) in a short period of timein order to prepare the subject for the trauma.

As should be clear, there is no requirement that any or all of die RICregimens in a repeated RIC regimen be identical with respect to timing,number of cycles per regimen, supra-systolic pressure, location, and thelike. Moreover, even within a regimen, the times of ischemia andreperfusion may differ between cycles. Typically, however, for ease ofuse, the cycles within a given regimen are identical. The inventionfurther contemplates that regimens performed prior to traumatic injurymay comprise more cycles and potentially may be more frequent and/orgreater in number than regimens performed during or following traumaticinjury.

In some instances, RIC may be performed in an area of the body that isremote to the area of traumatic injury. However, in some instances, theeffects of the traumatic injury are systemic (e.g., multi-organ injuryor dysfunction) and thus RIC is performed in an area of the body that isaccessible and preferably has not open wounds. Preferably but notexclusively, the RIC regimen is non-invasive. Accordingly, RIC may beperformed on a limb such as an upper or lower limb. The repeated RICregimen may be performed on a single site or on multiple sites in thebody. For example, the repeated RIC regimen may comprise a first RICregimen performed on the right upper arm, followed by a second RICregimen performed on the left upper arm. The repeated RIC regimen mayalternate between or cycle through sites on the body. In some instances,an RIC regimen may be performed on a subject at two different sites atoverlapping times including simultaneously. In such instances, twodevices may be used. These devices may be in communication with eachother or they may be functioning independently of each other.

The subjects of the invention will preferably be humans, althoughnon-human subjects such as companion animals (e.g., dogs, cats, etc.),agricultural or prize-winning animals (e.g., race horses, etc.) are alsocontemplated. Essentially, any subject that can experience traumaticinjury can be treated according to the invention.

Trauma, as used herein, refers to critical or severe bodily injury,wound or shock. These forms of trauma typically require some form ofresuscitation therapy. Resuscitation therapy typically involvesreplenishment of bodily fluids including but not limited to bloodtransfusion or other saline transfusion. Shock broadly refers tocirculatory dysfunction. Shock may be hemorrhagic or hypovolemic shock(associated with inadequate blood volume) or it may be cardiogenic shock(associated with inadequate output of blood from the heart). Traumaassociated with blood loss therefore typically also involves shock.Symptoms associated with shock include without limitation low bloodpressure (i.e., hypotension), hypovolemia, hyperventilation, andcyanotic skin. In some instances, the trauma involves traumatic braininjury (e.g., the injury is to the head). In some instances, the traumadoes not involve traumatic brain injury (e.g., the injury may be to thetorso or one or more limbs).

Therefore, RIC may be performed on a subject that is hypovolemic and/orhypotensive. A subject that is hypovolemic may be a subject that haslost 5%, 10%, 15%, 20%, 25%, 30% or more of its whole blood volume. Thecause of blood loss volume may be external bleeding, internal bleeding,or reduced blood volume resulting from excessive loss of other bodyfluids as may occur with diarrhea, vomiting and burns.

Trauma may result from direct injury such as penetrating injury (e.g.,bullet wound). Trauma may also result from indirect injury such as, forexample, a blast injury that occurs from exposure to a pressure wavefollowing, for example, an explosion. Such latter types of trauma mayoccur in the absence of hypovolemia. In some instances, the inventioncontemplates the use of RIC after traumatic injury not associated withhypovolemia. In these and other instances, RIC may diminish systemicmanifestations of the response to injury which includes neurologicinjury and multi-organ dysfunction.

Since it is important to treat the subject as soon as possible, theinvention contemplates that the methods provided herein may be performedin a hospital setting or in a non-hospital setting including in theenvironment in which the trauma occurred. RIC may be performed beforethe trauma occurs, and/or after the trauma occurs, including beforeand/or after resuscitation therapy is performed.

The repeated RIC regimens may be performed before, during and/or aftertrauma. In some embodiments, at least one of the RIC regimens areperformed before trauma. These are referred to as remote ischemicpreconditioning regimens. In these embodiments, at least one RIC regimenmay be performed within about 48 hours, within about 24 hours, withinabout 12 hours, within about 6 hours, within about 4 hours, within about2 hours, or within about 1 hour prior to trauma.

In some embodiments, at least one of the RIC regimens is performed aftertrauma has occurred. As an example, the subject may no longer byhypotensive or hypovolemic, but RIC may still be performed. These arereferred to as remote ischemic post-conditioning regimens. In theseembodiments, at least one RIC regimen may be performed within about 48hours, within about 24 hours, within about 12 hours, within about 6hours, within about 4 hours, within about 2 hours, within about 1 hour,within about 30 minutes, within about 20 minutes, within about 10minutes, within about 5 minutes, or just immediately after theoccurrence of trauma, or after the subject has been stabilized (e.g.,the subject is no longer hypovolemic and/or hypotensive).

In some embodiments, the repeated RIC regimens span a number of days,including 2, 3, 4, 5, 6, 7, 8, 9, 10,15, or 30 or more days. it is to beunderstood that in such instances, a subject may undergo an RIC regimendaily, or every 2, 3, 4, 5, or 6 days, for example. Additionally, theRIC regimens may be performed in a non-regular, or random, manner.

Additional Therapies

The repeated RIC regimen of the invention may be used in combinationwith other therapies or procedures aimed at treating traumatic injury,blood loss, hemorrhage, and/or shock. Depending upon the embodiment, oneor more of these agents may be administered before, simultaneously withor following one or more RIC regimens and/or before, simultaneously withor following trauma. Trauma victims may be administered an anaestheticsimply to alleviate pain associated. with the traumatic injury.

In some embodiments, the subject is administered a potassium channelopener or agonist. In some embodiments, the subject is administered anadenosine receptor agonist. In some embodiments, the subject isadministered both of the foregoing agents. Potassium channel openersinclude without limitation nicorandil, diazoxide, minoxidil, pinacidil,aprikalim, cromokulim and derivative U-89232, P-1075 (a selective plasmamembrane K-ATP channel opener), emakalim, YM-934,(+)-7,8-dihydro-6,6-dimethyl-7-hydroxy-8-(2-oxo-1-piperidinyl)-6H-pyrano[-2,3-f]benz-2,1,3-oxadiazole(NIP-121), RO316930, RWJ29009, SDZPCO400, rimakalim, symakalim, YM099,2-(7,8)-dihydro-6,6-dimethyl-6H-[1,4]oxazino[2,3-f][2,1,3]benzoxadiazol-8-yl)pyridine N-oxide,9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridinedione(ZM244085),[(9R)-9-(4-fluoro-3-125iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thi-eno[2,3-e]pyridin-8(7H)-one-1,1-dioxide]([125I]A-312110),(−)-N-(2-ethoxyphenyl)-N′-(1,2,3-trimethylpropyl)-2-nitroethene-1,1-diami-ne(Bay X 9228), N-(4-benzoylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamine (ZD6169), ZD6169(K-ATP opener) and ZD0947 (K-ATP opener), WAY-133537 and a noveldihydropyridine potassium channel opener, A-278637. In addition,potassium channel openers can be selected from BK-activators (alsocalled BK-openers or BK(Ca)-type potassium channel openers orlarge-conductance calcium-activated potassium channel openers) such asbenzimidazolone derivatives NS004(5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidaz-ole-2-one),NS1619(1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one),NS1608(N-(3-(trifluoromethyl)phenyl)-N′-(2-hydroxy-5-chlorophenyl)urea),BMS-204352, retigabine (also GABA agonist). There are also intermediate(e.g., benzoxazoles, chlorzoxazone and zoxazolamine) andsmall-conductance calcium-activated potassium channel openers. Othercompounds that are believed to open K-ATP channels include Levosimendanand hydrogen sulphide gas (H.sub.2S) or the H.sub.2S donors (eg sodiumhydmsulphide, NaHS).

Adenosine receptor agonists include without limitationN⁶-cyclopentyladenosine (CPA), N-ethylcarboxamido adenosine (NECA),2-[p-(2-carboxyethyl)phenethyl-amino-5′-N-ethylcarboxamido adenosine(CGS-21680), 2-chloroadenosine,N⁶-[2-(3,5-demethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine,2-chloro-N-6-cyclopentyladenosine (CCPR),N-(4-aminobenzyl)-9-[5-(methylcarbonyl)-beta-D-robofuranosyl]-adenine(AB-MECA),([IS-[1a,2b,3b,4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methyl-propyl]amino]-3H-imidazole[4,5-b]pyridyl-3-yl]cyclopentanecarboxamide: (AMP579), N⁶-(R)-phenylisopropyladenosine (R-PLA),aminophenylethyladenosine (APNEA) and cyclohexyladenosine (CHA).

Adenosine A1 receptor agonists include without limitationN-[3-(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT-510), or partialagonists such as CVT-2759 and allosteric enhancers such as PD81723.Other agonists may include N⁶-cyclopentyl-2-(3phenylaminocarbonyltriazene-1-yl) adenosine (TCPA).

In some embodiments, the subjects may be administered anti-inflammatoryagents, beta blockers (i.e., beta-adrenergic blocking agents) and/orcalcium channel blockers.

Anti-inflammatory agents include without limitation Alclofenac;Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase;Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride;Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium;Bendazac; Benoxaprofen; Benzydaniine Hydrochloride; Bromelains;Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;Clobetasol Propionate; Clobetasone Butyrate; Clopirac; CloticasonePropionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium;Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flutbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam.;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Salycilates; Sanguinarium Chloride; Seciazone; Sermetacin; Sudoxicam;Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone;Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine;Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Glucocorticoids; Zomepirac Sodium. Onepreferred anti-inflammatory agent is aspirin. vCalcium channel blockersare a chemically diverse class of compounds having important therapeuticvalue in the control of a variety of diseases including severalcardiovascular disorders, such as hypertension, angina, and cardiacarrhythmias (Fleckenstein, Cir. Res. v. 52, (suppl. 1), p. 13-16 (1983);Fleckenstein, Experimental Facts and Therapeutic Prospects, John Wiley,New York (1983); McCall, D., Curr Pract Cardiol, v. 10, p. 1-11 (1985)).Calcium channel blockers are a heterogeneous group of drugs that preventor slow the entry of calcium into cells by regulating cellular calciumchannels. (Remington, The Science and Practice of Pharmacy, NineteenthEdition, Mack Publishing Company, Eaton, Pa., p. 963 (1995)). Most ofthe currently available calcium channel blockers, and useful accordingto the present invention, belong to one of five major chemical groups ofdrugs. These are the dihydropyridines, such as nifedipine, nicardipine,and nimodipine; the phenyl alkyl amines, such as verapamil; thebenzothiazepines, such as diltiazem; the diarylaminopropylamine ethers,such as bepridil; and the benzimidazole-substituted tetralines, such asmibefradil.

Other calcium channel blockers useful according to the invention includebut are not limited to amrinone, amlodipine, bencyclane, felodipine,fendiline, flunarizine, isradipine, perhexilene, gallopamil, tiapamiland tiapamil analogues (such as 1993RO-11-2933), phenytoin,barbiturates, and the peptides dynorphin, omega-conotoxin, andomega-agatoxin, and the like and/or pharmaceutically acceptable saltsthereof.

Beta-adrenergic receptor blocking agents are a class of drugs thatantagonize the cardiovascular effects of catecholamines in anginapectoris, hypertension, and cardiac arrhythmias. Beta-adrenergicreceptor blockers include, but are not limited to, atenolol, acebutolol,alprenolol, befunolol, betaxolol, bunitrolol, carteolol, celiprolol,hedroxalol, indenolol, labetalol, levobunolol, mepindolol, methypranol,rnetindol, metoprolol, metrizoranolol, oxprenolol, pindolol,propranolol, practolol, practolol, sotalolnadolol, tiprenolol,tomalolol, timolol, bupranolol, penbutolol, trimepranol,2-(3-(1,1-dimethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHCl,1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminpropoxy)phthalide. The above-identifiedcompounds can be used as isomeric mixtures, or in their respectivelevorotating or dextrorotating form.

In some embodiments, the subjects may be administered antioxidantagents.

Antioxidants are agents that reduce or prevent damage associated withoxidation in tissue or organs. Antioxidants include without limitationallopurinol, carnosine, histidine, Coenzyme Q 10, n-acetyl-cysteine,superoxide dismutase (SOD), glutathione reductase (GR), glutathioneperoxidase (GP) modulators and regulators, catalase and the othermetalloenzymes, NADPH and AND(P)H oxidase inhibitors, glutathione,U-74006F, vitamin E, Trolox (soluble form of vitamin E), othertocopherols (gamma and alpha, beta, delta), tocotrienols, ascorbic acid,Vitamin C, Beta-Carotene (plant form of vitamin A), selenium, GammaLinoleic Acid (GLA), alpha-lipoic acid, uric acid (urate), curcumin,bilirubin, proanthocyanidins, epigallocatechin gallate, Lutein,lycopene, bioflavonoids, polyphenols, trolox(R), dimethylthiourea,tempol(R), carotenoids, coenzyme Q, melatonin, flavonoids, polyphenols,aminoindoles, probucol and nitecapone, 21-aminosteroids or lazaroids,sulphydryl-containing compounds (thiazolidine, Ebselen,dithiolethiones), and N-acetylcysteine.

Other antioxidants include the ACE inhibitors which are described ingreater detail below. Other antioxidants includebeta-mercaptopropionylglycine, O-phenanthroline, dithiocarbamate,selegiline, desferrioxamine (Desferal),5′-5-dimethyl-1-pyrrolione-N-oxide (DMPO) and(a-4-pyridyl-1-oxide)-N-t-butylnitrone (POBN). Other antioxidantsinclude nitrone radical scavenger alpha-phenyl-tert-N-butyl nitrone(PBN) and derivatives PBN (including disulphur derivatives);N-2-mercaptopropionyl glycine (MPG) a specific scavenger of the OH freeradical; lipooxygenase inhibitor nordihydroguaretic acid (NDGA); AlphaLipoic Acid; Chondroitin Sulfate; L-Cysteine; oxypurinol and. Zinc.

An angiotensin system inhibitor is an agent that interferes with thefunction, synthesis or catabolism of angiotensin II. These agentsinclude, but are not limited to, angiotensin-converting enzyme (ACE)inhibitors, angiotensin II antagonists, angiotensin II receptorantagonists, agents that activate the catabolism of angiotensin II, andagents that prevent the synthesis of angiotensin I from whichangiotensin II is ultimately derived. The renin-angiotensin system isinvolved in the regulation of hemodynamics and water and electrolytebalance. Factors that lower blood volume, renal perfusion pressure, orthe concentration of Na⁺ in plasma tend to activate the system, whilefactors that increase these parameters tend to suppress its function.

Angiotensin II antagonists are compounds which interfere with theactivity of angiotensin II by binding to angiotensin II receptors andinterfering with its activity. Angiotensin II antagonists are well knownand include peptide compounds and non-peptide compounds. Mostangiotensin H antagonists are slightly modified congeners in whichagonist activity is attenuated by replacement of phenylalanine inposition 8 with some other amino acid; stability can be enhanced byother replacements that slow degeneration in vivo. Examples ofangiotensin II antagonists include: peptidic compounds (e.g., saralasin,[(San¹)(Val⁵)(Ala⁸)] angiotensin -(1-8) octapeptide and relatedanalogs); N-substituted imidazole-2-one (U.S. Pat. No. 5,087,634);imidazole acetate derivatives including2-N-butyl-4-chioro-1-(2-chlorobenzile) imidazole-5-acetic acid (see Longet al., J. Pharmacol. Exp. Ther. 247(1), 1-7 (1988)); 4, 5, 6,7-tetrahydro-1H-imidazo [4, 5-c] pyridine-6-carboxylic acid and analogderivatives (U.S. Pat. No. 4,816,463); N2-tetrazole beta-glucuronideanalogs (U.S. Pat. No. 5,085,992); substituted pyrroles, pyrazoles, andtryazoles (U.S. Pat. No. 5,081,127); phenol and heterocyclic derivativessuch as 1, 3-imidazoles (U.S. Pat. No. 5,073,566); imidazo-fused7-member ring heterocycles (U.S. Pat. No. 5,064,825); peptides (e.g.,U.S. Pat. No. 4,772,684); antibodies to angiotensin II (e.g., U.S. Pat.No. 4,302,386); and aralkyl imidazole compounds such as biphenyl-methylsubstituted imidazoles (e.g., EP Number 253,310, Jan. 20, 1988); ES8891(N-morpholinoacetyl-(-1-naphthyl)-L-alanyl-(4, thiazolyl)-L-alanyl (35,45)-4-amino-3-hydroxy-5-cyclo-hexapentanoyl-N-hexylamide, SankyoCompany, Ltd., Tokyo, Japan); SKFI 08566 (E-alpha-2-[2-butyl-1-(carboxyphenyl) methyl] 1H-imidazole-5-yl[methylane]-2-thiophenepropanoic acid,Smith Kline Beecham Pharmaceuticals, PA); Losartan (DUP753/MK954, DuPontMerck Pharmaceutical Company); Remikirin (RO42-5892, F. Hoffman LaRocheAG); A₂ agonists (Marion Merrill Dow) and certain non-peptideheterocycles (G.D.Searle and Company).

ACE inhibitors include amino acids and derivatives thereof, peptides,including di- and tri-peptides and antibodies to ACE which intervene inthe renin-angiotensin system by inhibiting the activity of ACE therebyreducing or eliminating the formation of pressor substance angiotensinII. ACE inhibitors have been used medically to treat hypertension,congestive heart failure, myocardial infarction and renal disease.Classes of compounds known to be useful as ACE inhibitors includeacylmercapto and mercaptoalkanoyl prolines such as captopril (U.S. Pat.No. 4,105,776) and zofenopril (U.S. Pat. No. 4,316,906), carboxyalkyldipeptides such as enalapril (U.S. Pat. No. 4,374,829), lisinopril (U.S.Pat. No. 4,374,829), quinapril (U.S. Pat. No. 4,344,949), ramipril (U.S.Pat. No. 4,587,258), and perindopril (U.S. Pat. No. 4,508,729),carboxyalkyl dipeptide mimics such as cilazapril (U.S. Pat. No.4,512,924) and benazapril (U.S. Pat. No. 4,410,520), phosphinylaikanoylprolines such as fosinopril (U.S. Pat. No. 4,337,201) and trandolopril.

In some embodiments, the subject is administered HMG-CoA reductaseinhibitors. Examples include, but are not limited to, simvastatin (U.S.Pat. No. 4,444,784), lovastatin (U.S. Pat. No. 4,231,938), pravastatinsodium (U.S. Pat. No. 4,346,227), fluvastatin (U.S. Pat. No. 4,739,073),atorvastatin (U.S. Pat. No. 5,273,995), cerivastatin, and numerousothers described in U.S. Pat. Nos. 5,622,985, 5,135,935, 5,356,896,4,920,109, 5,286,895, 5,262,435, 5,260,332, 5,317,031, 5,283,256,5,256,689, 5,182,298, 5,369,125, 5,302,604, 5,166,171, 5,202,327,5,276,021, 5,196,440, 5,091,386, 5,091,378, 4,904,646, 5,385,932,5,250,435, 5,132,312, 5,130,306, 5,116,870, 5,112,857, 5,102,911,5,098,931, 5,081,136, 5,025,000, 5,021,453, 5,017,716, 5,001,144,5,001,128, 4,997,837, 4,996,234, 4,994,494, 4,992,429, 4.970,231,4,968,693, 4,963,538, 4,957,940, 4,950,675, 4,946,864, 4,946,860,4,940,800, 4,940,727, 4,939,143, 4,929,620, 4,923,861, 4,906,657,4,906,624 and 4,897,402, the disclosures of which patents areincorporated herein by reference.

It is to be understood that the invention contemplates the use of one ormore of any of the foregoing agents in combination with RIC of theinvention.

RIC

As used herein, a RIC regimen is at least one cycle of an inducedtransient ischemic event followed by a reperfusion event. Typically,these regimens are performed by restricting blood flow in a limb or aperipheral tissue of the subject and then removing the blood flowrestriction and allowing blood to reperfuse the limb or tissue. Aregimen may comprise a single cycle or multiple cycles, including 2, 3,4, 5, or more cycles. In one important embodiment, a regimen comprises 4cycles of ischemia and reperfusion.

The blood flow restriction typically takes the form of an appliedpressure to the limb or tissue that occludes blood flow. The appliedpressure to the limb or tissue may be above systolic pressure (i.e.,supra-systolic pressure). It may be about 5, about 10, about 15, about20, or more mmHg above (or greater than) systolic pressure. Sincesystolic pressure will differ between subjects, the absolute pressureneeded to induce ischemia will vary between subjects. In otherembodiments, the pressure may be preset at, for example, 200 mmHg. Insome embodiments, the applied pressure is less than systolic pressureprovided blood flow is occluded. It is to be understood that, as usedherein, blood flow occlusion refers to cessation of blood flow. Asdescribed herein, such blood flow occlusion or cessation occurs in aregion of the body that is remote from a site of injury or one or moreorgans being protected from such injury. For example, blood flowocclusion or cessation may occur in a lower limb and/or an upper limb.

The blood flow restriction or occlusion may be accomplished using anymethod as the invention is not limited in this regard. Typically, it maybe accomplished with an inflatable cuff, although a tourniquet system isalso suitable. Further examples of automated devices for performing RICare described below.

The induced ischemic event is transient. That is, it may have a durationof about 1, about 2, about 3, about 4, about 5, or more minutes.Similarly, the reperfusion event may have a duration of about 1, about2, about 3, about 4, about 5, or more minutes.

If performed using a limb, the upper limb or lower limb may be used. Insome instances the upper limb is preferred. In some instances the lowerlimb is preferred. In some instances, RIC is performed on two differentsites on the body, in an overlapping or simultaneous mariner.

RIC may be performed using any device provided it is capable of inducingtransient ischemia and reperfusion, whether manually or automatically.

In one of its simplest forms, the method may be carried out using asphygmomanometer (i.e., the instrument typically used to measure asubject's blood pressure). The cuff of the sphygmomanometer is placedabout a subject's limb (e.g., an arm or leg) and is inflated to apressure great enough to occlude blood flow through the limb (i.e., apressure greater than the subject's systolic blood pressure). The cuffis maintained in the inflated state to prevent blood flow through thelimb for a specified period of time, referred to herein as the ischemicduration. After the ischemic duration, pressure is released from thecuff to allow reperfusion of blood through the limb for a period of timethat is referred herein as the reperfusion duration. The cuff is thenre-inflated and the procedure is immediately repeated a number of times.

The method may similarly be carried out using a manual type tourniquet.Devices such as those described in published PCT application WO 83/00995and in published US application 20060058717 may also be used.

Another system that may be used is described in published US application20080139949. The advantage of this system is that it can be usedindependently of a medical practitioner, and that it automatically'induces the required RIC regimen. This system is exemplified in part inFIG. 1, which illustrates a cuff 10, an actuator 12, a controller 14 anda user interface 16. The cuff is configured to be placed about the limb15 of a subject, such as an arm or leg of the subject. The actuator,when actuated, causes the cuff to retract about the limb to occludeblood flow through the limb. The controller executes a protocol thatcomprises repeating a cycle one or more times. The cycle itself includesactuating the cuff to prevent blood flow, maintaining the cuff in anactuated state for an ischemic duration, releasing the cuff, andmaintaining the cuff in a relaxed state to allow reperfusion.

FIG. 2 shows a block diagram that represents a scheme that may be usedto perform RIC. The scheme begins with placement of a cuff about asubject's limb. The system is then activated and the protocol isinitiated through the controller. In one embodiment, the system isactivated by a medical professional. In another embodiment, the systemmay be activated by the subject. The cuff contracts to apply an initialpressure, greater than systolic pressure, to the subject's limb. Asdiscussed herein, the initial pressure may be a default value of thesystem or may be programmed into a particular protocol. The cuff thendeflates to identify the subject's systolic pressure. This may beaccompanied by monitoring the subject for the onset of Korotkoff soundsor vibrations. Alternatively or additionally, a distal remote sensor(e.g., a device on the fingertip which is sensitive to the presence orabsence of flow or maintenance of flow) may be used. Once systolicpressure has been identified, the system initiates the first cycle ofthe protocol. n some embodiments, systolic pressure may be identified asan initial portion of the protocol. As used herein, the terms protocoland regimen are used interchangeably.

The cycle begins as the cuff contracts to apply a target pressure,greater than the subject's systolic pressure by an amount defined in theprotocol, to the subject's limb. This occludes blood flow through thesubject's limb. The external pressure against the subject's limb is heldfor an ischemic duration defined in the protocol. The system monitorsthe subject during the ischemic duration for pressure release criteria,which may include system power failure, system power spikes, and manualactivation of quick release mechanism, The system also monitors thesubject during the ischemic duration for any signs of reperfusionthrough the subject's limb, and accordingly, increases the externalpressure applied by the cuff to prevent such reperfusion. Signs ofreperfusion can include the onset of Korotkoff sounds or vibrations.After passage of the ischemic duration, the cuff releases pressure fromabout the subject's limb to allow reperfusion. Reperfusion is allowedfor a reperfusion duration defined in the cycle.

The initial cycle typically concludes after the reperfusion duration. Atthis time, a subsequent cycle may begin as the cuff is actuated tocontract about the subject's limb to occlude blood flow through the limbfor another ischemic duration.

The cuff illustrated in FIG. 1 is configured to be positioned about thelimb of a subject and to contract about the limb when actuated. In oneembodiment, the sleeve is wrapped about a subject's upper arm, calf, orthigh and is fastened snugly in place. Portions of the cuff may includehook and loop type material that can be used to fasten the sleeve inplace about the subject's limb. The actuator inflates the cuff such thatthe limb is constricted to the point of occluding blood flow through thesubject's limb.

The illustrated cuff includes an inflatable bladder (not shown) thatreceives a fluid, such as air, to cause the cuff expand and retractabout a subject's limb. The bladder is constructed of an air impermeablematerial, such as flexible plastic or rubber. A connection port 18 ispresent at one end of the bladder to allow air to enter the bladderduring inflation, or to exit the bladder during deflation. The port mayinclude engagement features to facilitate a connection to the actuator,such as by an air hose. These features may include threads, clips, andthe like. Although the illustrated embodiment includes a single bladderpositioned within a cuff, it is to be appreciated that other embodimentsare also possible. By way of example, according to some embodiments, thefabric sleeve may itself be air impermeable, such that no separatebladder is required. In other embodiments, multiple, separate inflatablebladders may be incorporated into a common sleeve, as aspects of thepresent invention are not limited in this respect.

The general size of subjects that undergo RIC may vary greatly,particularly given the range of species to which the methods may beapplied. Given this variance, it may be desirable for some embodimentsof cuffs to be adjustable over a wide range to accommodate the varietyof subject limb girths that may be expected. According to someembodiments, the cuff comprises an inflatable fabric sleeve having alength greater than three feet, such that a girth of up to three feetmay be accommodated. Embodiments of cuffs may include a width as smallas two inches, one inch, or even smaller, so as to accommodate the upperarm or leg of a much smaller subject, including a neonatal infant. It isto be appreciated, however, that other embodiments may be configured toencircle a much smaller range of limb sizes, as aspects of the presentinvention are not limited in this regard.

Various devices may be used as an actuator to constrict the cuff about asubject's limb, or to release the cuff. As illustrated in embodiment ofFIG. 1, the actuator includes a pneumatic pump to provide pressurizedair to an inflatable cuff through an air hose. The actuator alsoincludes a release valve 20 that, when actuated, opens a passagewaybetween the inflatable cuff and the external environment to allowpressurized air to escape from the cuff, so that the cuff loosens aboutthe subject's limb.

The air pump can comprise any device capable of delivering compressedair. According to some embodiments, the air pump includes a pistoncompressor, although other types of pumps, like centrifugal pumps andscroll compressor may also be used. The pump may be configured toprovide air flow at a rate of between 0.1 to 20 cubic feet per minute,with a head pressure of up to 50 psi, according to some embodiments.However, other flow rates and/or pressures are possible, as aspects ofthe invention are not limited in this respect.

The device may therefore comprise or be suited for use with a compressedair canister or cartridge. Pressurized gas (e.g., air) can then be usedto inflate the band, thereby constricting the limb. In certainsituations, including situations in which traumatic injury is common orlikely (e.g., on a battlefield), the use of compressed gas may be moresuitable than other inflation or constriction mechanisms. As an example,compressed gas canisters or cartridges are less likely to become cloggedor otherwise fouled by debris that may be encountered (e.g., such asmud, dirt, water, etc. that exists in battlefield environments)

As discussed above, the actuator may also include a release mechanism torelease a cuff from about the subject's limb. In the illustratedembodiment, the release comprises a release valve 20 that is positionedwithin the controller housing. The release valve, as shown, may be asolenoid that moves rapidly between fully closed and fully openpositions to rapidly release air from the cuff and, in turn, to rapidlyrelease the cuff from a subject. According to some embodiments, the samerelease valve or another release valve may also be actuated to openslowly, such as to adjust the pressure of the cuff or to allow a morecontrolled release of pressure such as may be required when thesubject's blood pressure is measured.

Embodiments of the system may include safety features to allow rapidrelease of the cuff from a subject's limb. Moreover, some of theseembodiments may be readily activated by a subject, such as when thesubject feels discomfort. In one embodiment, the safety release 22includes a large button positioned on or near the cuff. In this regard,the safety release is within reach of the subject. in other embodiments,the safety release may comprise a separate actuator, such as one thatmay be held in the free hand of the subject. Activating the safetyrelease may cause the release valve of a pneumatic cuff to open, therebyallowing rapid removal of air from the cuff.

The system may also include a continually operating, cuff releasemechanism. By way of example, a slow release valve may be incorporatedinto a pneumatic cuff to provide for a continual, slow release ofpressurized air from the cuff. The continual slow release mechanism mayprovide for the safe release of a subject's limb, even in the face ofpower failures or other events that may prevent redundant safetyfeatures from operating properly. Similar type mechanism may beincorporated into embodiments that do not utilize a pneumaticallyinflatable cuff, as continual slow release mechanisms are not limited topneumatic cuffs.

Embodiments of the system include a controller that receives informationfrom a protocol and any other sensors in the system to, in turn, controlthe actuator to perform RIC. The controller and protocol combination maybe implemented in any of numerous ways. For example, in one embodimentthe controller and protocol combination may be implemented usinghardware, software or a combination thereof. When implemented insoftware, the software code can be executed on any suitable processor orcollection of processors, whether provided in a single computer ordistributed among multiple computers. It should be appreciated that anycomponent or collection of components that perform the functionsdescribed herein can be generically considered as one or morecontrollers that control the functions discussed herein. The one or morecontrollers can be implemented in numerous ways, such as with dedicatedhardware, or with general purpose hardware (e.g., one or moreprocessors) that is programmed using microcode or software to performthe functions recited above. The one or more controllers may be includedin one or more host computers, one or more storage systems, or any othertype of computer that may include one or more storage devices coupled tothe one or more controllers. In one embodiment, the controller includesa communication link to communicate wirelessly, or via electrical oroptical cable, to a remote location.

In this respect, it should be appreciated that one implementation of theembodiments of the present invention comprises at least onecomputer-readable medium (e.g., a computer memory, a floppy disk, acompact disk, a tape, etc.) encoded with a protocol in the form of acomputer program (i.e., a plurality of instructions), which, whenexecuted by the controller, performs the herein-discussed functions ofthe embodiments of the present invention. The computer-readable mediumcan be transportable such that the protocol stored thereon can be loadedonto any computer system resource to implement the aspects of thepresent invention discussed herein. In addition, it should beappreciated that the reference to a protocol or controller which, whenexecuted, performs the herein-discussed functions, is not limited to anapplication program running on a host computer. Rather, the termprotocol is used herein in a generic sense to reference any type ofcomputer code (e.g., software or microcode) that can be employed toprogram a processor to implement the herein-discussed aspects of thepresent invention.

The system may also comprise one or more sensors 26 that receiveinformation from the subject and/or portions of the system itself. Suchsensors may receive information regarding blood flow in any portion ofthe subject, including the limb that is being treated. These sensors mayalso receive information regarding other operating parameters of thesystem, such as air pressure within a pneumatic cuff, direct readings ofpressure applied by cuff, or tension within portions of a tension band.

Pneumatic cuffs may include a sensor to measure pressure within thecuff. Cuff pressure is often directly indicative of the pressure thatexists within a blood vessel of the limb beneath the cuff. Thecontroller of a system is often programmed to target a particular cuffpressure that is to be maintained during the ischemic duration of acycle, as is discussed herein. In embodiments that include a pneumaticcuff, the pressure sensor may be positioned anywhere within thepressurized space of the cuff, the air hose, or even within the actuatoritself. Pressure sensors may also be positioned on an inner surface ofthe cuff to directly measure the pressure, between the cuff and an outersurface of the subject's limb. In use, the cuff may be oriented suchthat the pressure sensor is positioned directly above the subject'sartery, so as to provide a more direct measurement of pressure at ablood vessel of interest.

In one embodiment, systems may also include one or more vibration and/orultrasonic sensors 28 to identify Korotkoff sounds. Korotkoff sounds aregenerally understood to be present when pressures between systolic anddiastolic are externally applied to the artery of a subject. Systolicpressure is associated with a pressure value that completely occludesblood flow through a subject's blood vessels, and in this regard, may beused by the system as feedback to identify when pressure in the systemis low enough to allow blood flow, or high enough to occlude blood flow.

One or more sensors may be included to confirm the cessation of bloodflow or reperfusion in the limb that receives the cuff. For instance, insome embodiments, a pulse oximeter 30 may be positioned on a distalportion of the limb that receives the cuff, such as on a finger or toeof the limb. The pulse oximeter can provide information regarding bloodpulsing through the subject's blood vessels and the percentage ofhaemoglobin that is saturated with oxygen. The pulse oximeter willdetect an absence of pulses when blood flow though a limb is notoccurring to confirm the occlusion of blood flow. Moreover, the pulseoximeter may also detect the percentage of haemoglobin saturated withoxygen, which will drop as blood flow through the limb ceases. It is tobe appreciated that other sensors may also be used to confirm thecessation of blood flow, such as a photoplethysmographic transducer, anultrasonic flow transducer, a temperature transducer, an infrareddetector, and a near infrared transducer, as aspects of the inventionare not limited in this respect.

As mentioned above, the system includes a protocol that, through thecontroller, directs the operation of the system. Embodiments of theprotocol include a cycle that comprises cuff actuation, an ischemicduration, cuff release, and a reperfusion duration. In many embodimentsof protocols, the cycle may be repeated multiple times. Additionally,sonic embodiments of the protocol include systolic pressureidentification.

The cuff actuation portion of the cycle comprises contracting the cuffabout the limb of a subject to occlude blood flow through the limb.Contraction of the cuff is accomplished by the controller readinginstructions from the protocol, such as a target set point for cuffpressure, and then by the initiating the controller to bring the cuff tothe target set point. Attainment of the target set point may be sensedthrough any of the herein described sensors and techniques.

During the ischemic phase of the cycle, pressure is maintained about thesubject's limb to prevent reperfusion of blood flow through the limb.The length of the ischemic phase, termed the ischemic duration, istypically defined by a doctor, or other medical professional, and isprogrammed into the protocol. Ischemic duration may be as short as a fewseconds, or as long as 20 minutes, or even longer, as aspects of theinvention are not limited in this regard. In some embodiments, theischemic duration varies from cycle to cycle during the same protocol,although in other embodiments, the ischemic duration remains constant.

The controller acts to maintain pressure, applied by the cuff, at a setpoint above the subject's systolic pressure. Embodiments of the cuff mayrelax relative to the subject's limb over time, thereby reducingpressure and eventually allowing reperfusion. This may be caused byvarious factors, including relaxation of muscles in the subject's limb,stretching of the cuff about the limb, air leaks (intentional orunintentional), and the like. To this end, a sensor may provide pressurereadings as feedback to the controller. The controller can measure anydifference between the set point and the actual pressure reading and canprovide any necessary commands to the actuator to compensate for errors.

Various approaches may be used to define an appropriate set point forthe controller during the ischemic duration. According to oneembodiment, the set point is manually entered into the protocol by thedoctor (or other medical professional). Alternately, the doctor mayselect a set point in terms of the subject's systolic blood pressure. Inone embodiment, the set point may be selected as a fixed pressure amountover the subject's systolic blood pressure, such as 5 mmHg, 10 mmHg, 15mmHg, 20 mmHg, 25 mmHg, 30 mmHg, or any other fixed amount abovesystolic pressure of the subject. In other embodiments, the set pointmay be defined as a percentage of the subject's systolic blood pressure,such as 102% of systolic, 105%, 110%, 115%, and other percentages, asaspects of the invention are not limited in this respect. The pointabove systolic pressure may be set by the medical professional and maybe dependent upon several factors including, but not limited to the sizeof the subject, the size of the subject's limb, the subject's bloodpressure, confirmation of blood flow cessation, and the like. In stillother embodiments, the pressure may be set below systolic pressure, suchas but not limited to 95%, 96%, 97%, 98%, or 99% of systolic pressure,provided that blood flow is occluded at such pressure, as may beachieved by varying cuff parameters.

The protocol, according to some embodiments, includes phases to identifythe subject's systolic blood pressure. The cuff may be allowed to loosenabout the subject's limb, from a point believed to be above systolicpressure, in a systematic manner while sensors are monitoring the limbfor the onset of Korotkoff sounds or vibrations. Once the systolicpressure is identified, the protocol may continue in the normal course.

Identification of systolic pressure may optionally occur at any timeduring a protocol, or not at all. According to some embodiments, eachcycle begins with the identification of the subject's systolic bloodpressure. In other embodiments, systolic pressure may be identified onlyonce during an initial portion of the protocol. In still otherembodiments, systolic pressure may be identified as the cuff is releasedduring the cuff release portion of each cycle. Still, as discuss herein,systolic pressure may not be identified at all during a protocol, asaspects of the invention are not limited in this regard.

The system can be configured to adjust the pressure set point during theischemic duration. As discussed herein, the system may include sensorsthat detect the onset of reperfusion. As an example, this may beaccomplished by detecting the presence of Korotkoff sounds orvibrations. The presence of Korotkoff sounds during an ischemic durationcan indicate that either cuff pressure has fallen below systolic or thatsystolic pressure has risen above the set point that was previouslyabove systolic pressure. Other devices may additionally or alternativelybe used including for example devices on digits that detect the presenceor absence of flow. In such a situation, the controller may adjust theset point based on the newly identified systolic pressure and/or otherinformation and in this regard, can identify and prevent unwantedreperfusion that might otherwise occur.

The cuff release portion of a cycle occurs at the end of the ischemicduration and. includes release of the cuff to a point below diastolicpressure. According to some embodiments, cuff release comprisesreleasing the pressure or tension of the cuff. In embodiments thatutilize a pneumatic cuff, this may simply be associated with moving anair release valve to the fully open position to allow a rapid reductionin cuff pressure and a corresponding rapid relaxation of the cuff aboutthe subject's limb. However, it is to be appreciated, that in otherembodiments, that cuff relaxation may occur in a slower, more controlledmanner, as aspects of the invention are not limited in this respect.Additionally, as discussed herein, the cuff release may be accompaniedby monitoring for the onset of Korotkoff sounds or vibrations toidentify or confirm the systolic pressure of the subject.

The reperfusion duration follows the cuff release in embodiments of thecycle. Reperfusion through the limb is allowed for a period of timetermed die reperfusion duration. Much like the ischemic duration,reperfusion may be allowed for varied lengths of time, as short as afive seconds, one minute or more, and as long as 20 minutes, or evenlonger. The reperfusion duration may remain constant from cycle to cycleduring a common protocol, or may vary between each cycle, as aspects ofthe invention are not limited in this respect.

The protocol may comprise any number of cycles. As discussed herein, acommon cycle may simply be repeated a plurality of times, such as two,three, four, or more times, to complete a protocol. Alternately, thecycles of a protocol may be programmed with different parameters, suchas different ischemic durations, reperfusion durations, pressure setpoints during the ischemic duration, and the like.

In some embodiments, the system may include a data logging feature thatrecords the system parameters, such as cuff pressure or tension, duringall phases of a protocol. Date of time of operation may also berecorded. Other features, such as personal information to identify thesubject, may also be recorded by the system.

Embodiments of the system may incorporate various features to inform thesubject or medical professional about the progress of the protocol.Audible or visual indicators may accompany any of the phases of theprotocol. By way of example, a clock may show either the amount of timethat has elapsed or that remains for a given portion of the protocol orthe entire protocol. Embodiments may also include other features to keepthe subject and/or medical professional informed, as aspects of theinvention are not limited in this regard.

According to some embodiments, the system includes features to preventtampering or accidental reprogramming by a subject. By way of example,in some embodiments, the reprogrammable features may only be accessedafter entering a code. This can prevent a subject from mistakenlyreprogramming the protocol or otherwise interfering with the operationof the system. It is to be appreciated that other devices may also beused to prevent accidental reprogramming, such as electronic keys,mechanical locks and the like.

The system may be configured for use is a variety of environments. Byway of example, the system may be mounted on a portable stand withcasters to facilitate easy movement. The stand may position thecontroller, user interface, and connections to the cuff at a convenientheight for the subject. In other embodiments, the system is configuredfor portable use. In such embodiments, the system may be configured forready placement into a suitcase for easy transport.

The system is also not limited to components illustrated in theembodiment of FIG. 1. By way of example, according to other embodiments,like that illustrated in FIG. 3, cuffs may be configured to constrict asubject's limb through alternative mechanisms. In the illustratedembodiment, the cuff is configured as a band having a ratchetingmechanism positioned at one end. In use, the band is wrapped about thelimb of a subject with the free end of the band passing through theratcheting mechanism. In such an embodiment, the actuator may comprise amechanism that pulls the free end of the hand further through theratcheting mechanism to retract the cuff about the limb, or that freesthe ratcheting mechanism to release the band to, in turn, release theband from the limb. Still other mechanisms, such as tourniquetmechanisms, are possible, as aspects of the invention are not limited inthis respect.

As described above with reference to FIG. 3, some embodiments may have acuff that comprises a hand that does not inflate, but rather istightened about a subject's limb by another mechanism. In suchembodiments, the actuator may comprise a tensioning mechanism configuredto move one end of the band relative to other portions of the band so asto place the band in tension. As shown, the mechanism can includeopposed rollers held in close proximity to one another within a housing.The housing includes a slot for receiving a free end of the band and afixation point for fixed attachment to the opposite end of the band. Thefree end of the band is passed into the slot and between the rollers.The rollers may be mechanically actuated to rotate relative to oneanother, such as by an electric motor, to pull the free end through thehousing and thus tighten the band around a subject's limb.

The tensioning mechanism may include opposed rollers mounted on aratcheting, free wheel mechanism. The freewheel mechanism allows theband to be pulled through the slot in one direction with minimalresistance so that the band may be pulled rapidly to a snug positionabout a subject's limb. The free wheel mechanism also prevents the bandfrom moving through the slot in the loosening direction, unless themechanism is released or the opposed rollers are actuated. It is to beappreciated that not all embodiments will include a tree wheelmechanism, as aspects of the invention are not limited in this regard.

The opposed rollers rotate in either direction to tighten and loosen theband during use. When required, the rollers may rapidly rotate until theband achieves a particular tension. The rollers may further be actuatedto make minor adjustments to the tension in the band during use. Whenthe cuff is to be released from the subject's limb, a ratchetingmechanism or clutch may be released such that the opposed rollers areallowed to move freely, thus rapidly releasing tension.

The invention contemplates the placement of a cuff or cuff-containingdevice such as those described herein within a garment, including butnot limited to a military uniform. The cuff may be placed in a pant orleg portion or in the sleeve or arm. portion of the garment. The garmentmay be designed such that the wearer is able to initiate one or morecycles of blood flow occlusion and reperfusion. The device may becapable of remote operation in the event that the wearer is unable toinitiate the occlusion/reperfusion cycles.

Aspects of the invention are not limited to the embodiments of cuffsillustrated herein.

EXAMPLES

Resuscitated hemorrhagic shock following trauma is known to contributeto the development of late organ dysfunction in those who survive theinitial trauma insult and thus contributes to morbidity and mortality inthis patient population. A number of mechanisms have been implicated inthis process, but the activation of proinflammatory signaling cascadesleading to cellular death directly via apoptotic pathways oralternatively through priming of the innate immune cell system have beenshown to be contributory. Strategies directed at preventing the onset ofthese pathways would clearly have potential benefit in reducingattendant morbidity and mortality related to organ injury.

Remote ischemic conditioning (RIC), a process whereby distant vascularbeds are temporarily rendered ischemic, protects organs from the effectsof ischemia/reperfusion (I/R) injury. For example, transient remotevascular occlusion has been shown to be protective of injury in theheart, liver, lung, intestine and kidney. This Example shows the effectof RIC on end organ injury, particularly in the lung and liver,following hemorrhagic shock.

Materials and Methods:

Animal model of hemorrhagic shock (HS). Animals were cared for inaccordance with the guidelines set forth by the Canadian Council onAnimal Care. C57B1/6 mice weighing 20 to 30 g (Charles River, StConstant, Quebec) were anaesthetized with intra-peritoneal ketamine (200mg/kg) and xylazine (10 mg/kg). The right femoral artery was cannulatedfor hemorrhagic shock and resuscitation. Hemorrhagic shock was initiatedby blood withdrawal equivalent to 20% of blood volume (22.5 mL ofblood/kg) over 15 mins as previously reported. To prevent clotting, shedblood was collected in the presence of 3.8% Na-citrate. After ahypotensive period of 1-10 mins or 60 mins, animals were resuscitated bytransfusion of the shed blood plus an equal volume of Ringer's Lactateusing a timed-delivery pump system. Total resuscitation time wasstandardized to two hours. Sham animals were instrumented but not bled.Finally, animals were sacrificed at the end of the resuscitation periodby pentobarbital overdose.

At the end of the resuscitation period (t=0) and at t=2, 4 and 6 hlater, a tracheostomy was performed and alveolar cells were recovered bybronchoalveolar lavage (BAL). In some studies, cell count anddifferential were performed following Giemsa staining and in otherstudies lung were harvested for histopathology and determination ofwet/dry ratios as a measure of lung injury. Cells recovered from BAL atthe end of a resuscitation period have been reported to exhibit a primedphenotype (Rizoli et al. J Immunol 61(11):6288-6296, 1998). Cellsrecovered at t=0 following resuscitated HS were plated and thenstimulated in vitro with LPS (100 ng/ml). Supernatants were recoveredfor measurement of TNF-α. by ELISA. In some experiments, the pelletedcells were re-suspended in serum-free DMEM and processed forimmunofluorescence staining or flow cytometry analysis. Primed cellshave also been reported to exhibit augmented Toll-like receptor 4 ontheir plasma membrane as a measure of priming (Powers et al. J Exp Med203(8):1951-1961, 2006).

Serum alanine aminotransferase (ALT) levels, an indicator ofhepatocellular injury, were measured in blood samples obtained atdifferent times after the end of the resuscitation period. Measurementsof ALT were made using BIOTRON Diagnostic Kits. Liver tissue was fixedby immersion in formaldehyde, embedded by paraffin wax, and then cut in5 μm slices. Sections were evaluated by light microscopy followingstaining by haematoxylin and eosin. TUNEL staining for apoptotic nucleiwas detected in dewaxed sections using die DeadEnd Fluorometric TUNELKit (Promega Corp).

RIC Protocol. Animals were subjected to unilateral hindlimb ischemia (orsham operation) using an elastic band around the thigh for varying timesand then released for an equivalent time prior to initiation ofhemorrhagic shock. Animals were subjected to 10 min of ischemia followedby 10 min of reperfusion.

Statistical Analysis. The data are presented as mean ±standard error ofn determinations as indicated in the Figure legends. Data were analyzedby one-way analysis of variance, and Newman-Keuls Multiple ComparisonTest post-hoc using the Prism software (GraphPad, San Diego, Calif.).Results were considered significant when p≤0.05. Significance isindicated with an asterisk.

Results:

FIG. 4 shows the effect of RIC on shock-resuscitation induced liverdamage as indicated by ALT levels in the treated subjects. FIG. 4A showsthe results from individual subjects while FIG. 4B shows the results asthe mean +/− standard error. The animals were divided into five groupsas follows:

(i) basal, in which animals were treated as described above for sham;

(ii) SIR—0′, in which animals were subjected to shock (15 mins of bloodwithdrawal) and then resuscitated within 0-10 minutes;

(iii) SIR—0′+Hind Limb, in which animals were ischemically conditionedprior to shock, then subjected to shock, and then resuscitated within1-10 minutes;

(iv) SIR—1 HR, in which animals were subjected to shock and thenresuscitated 60 minutes later; and

(v) S/R—1 HR+Hind. Limb, in which animals were ischemically conditionedprior to shock, then subjected to shock, and then resuscitated 60minutes later.

FIG. 4 shows that, on average, RIC prevents liver damage, as measured byALT levels, in subjects that have experienced hemorrhagic shock. RICreduces and/or prevents liver damage in subjects that receiveresuscitation therapy immediately following shock and in subjects thatreceive delayed resuscitation therapy.

FIG. 5 shows the effect of RIC on shock-resuscitation induced TNF-alphaserum levels. TNF-alpha is a mediator of systemic inflammation and canbe used as an indicator of liver damage. FIG. 5A shows the results fromindividual subjects while FIG. 5B shows the results as the mean +/−standard error. The animals were divided into five groups as describedabove for FIG. 4.

FIG. 5 shows that RIC prevents the induction of TNF-alpha levelsfollowing shock and resuscitation. In subjects that received RIC andwere resuscitated within 0-10 minutes of shock, serum TNF-alpha wasundetectable, as compared to an average level of 6.9 pg/ml in subjectsthat did not receive RIC. In subjects that received RIC and wereresuscitated 60 minutes after shock, serum TNF-alpha level was reducedalmost 25 fold, on average, as compared to subjects that did not receiveRIC. The level of serum TNF-alpha in subjects receiving RIC and delayedresuscitation more closely resembled that of sham (untreated) subjects.

Accordingly, the data show that RIC reduces or prevents injury resultingfrom shock, whether the subjects receive immediate or delayedresuscitation therapy.

Conclusion:

RIC represents a safe and effective approach to minimizing organ injuryin subjects at risk for hemorrhagic shock. These findings have relevanceto the preoperative surgical setting as well as civilian and militarytrauma injury.

The foregoing written specification is considered to be sufficient toenable one ordinarily skilled in the art to practice the invention. Thepresent invention is not to be limited in scope by examples provided,since the examples are intended as mere illustrations of one or moreaspects of the invention. Other functionally equivalent embodiments areconsidered within the scope of the invention. Various modifications ofthe invention in addition to those shown and described herein willbecome apparent to those skilled in the art from the foregoingdescription. Each of the limitations of the invention can encompassvarious embodiments of the invention. It is, therefore, anticipated thateach of the limitations of the invention involving any one element orcombinations of elements can he included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth orillustrated in the drawings. The invention is capable of otherembodiments and of being practiced or of being carried out in variousways.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including”, “comprising”, or “having”, “containing”, “involving”, andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

What is claimed is:
 1. A method for reducing or preventing injury tocells, tissues, or organs of a body as a result of trauma comprisingperforming individual or repeated remote ischemic conditioning (RIC)regimens on a subject before, during, and/or following trauma.
 2. Amethod for treating a subject experiencing trauma comprising performingindividual or repeated remote ischemic conditioning (RIC) regimens on asubject during and/or following trauma.
 3. A method comprisingperforming individual or repeated remote ischemic conditioning (RIC)regimens on a subject experiencing or likely to experience trauma,wherein the one or more RIC regimens are performed before, during and/orfollowing trauma. 4-30. (canceled)